The goal of this work is to understand the biosynthetic pathways of extracellular glycans in the encapsulated fungus Cryptococcus neoformans. This opportunistic pathogen is responsible for life threatening disease, particularly in the context of compromised immunity, and current therapy is not adequate. The main virulence factor of C. neoformans is an extensive polysaccharide capsule which is composed of two polymers, glucuronoxylomannan (GXM) and galactoxylomannan (GalXM). The structures of these polysaccharides have been chemically defined, but their biosynthesis is not understood. The proposed work addresses this gap in knowledge. In the preceding period of this project we succeeded in purifying, cloning, and expressing a novel mannosyl transferase (Cmt1p), and examining its role in GXM synthesis. The current application proposes further investigations of the biochemical properties and interacting partners of this enzyme (Aim I). Broadening the investigations to other activities required for capsule synthesis, a xylosyltransferase recently discovered by the investigator will also be purified and characterized (Aim II). Finally, the events required for binding of capsule to the surface of the cryptococcal cell will be investigated, building on observations by the investigator and her group that cell wall alpha glucan synthase is required for this process (Aim III). The broad, long-term objective of the proposed research is to investigate novel features of C. neoformans glycobiology, which will lead to greater understanding of cryptococcal biology, identification of targets for anti-fungal drug discovery, and elucidation of glycan synthesis in eukaryotes. [unreadable] [unreadable] [unreadable]